Method of determining global coagulability and hemostatic potential

ABSTRACT

A method is disclosed for determining if a patient is hypercoagulable, hypocoagulable or normal. The test involves providing a test sample from the patient and initiating coagulation in the sample in the presence of an activator, which is added to the sample in an amount which will result in intrinsic tenase-dependent fibrin. Then the formation of the intrinsic tenase-dependent fibrin polymerization is monitored over time so as to derive a time-dependent profile, with the results of the fibrin polymerization monitoring determining whether the patient is hypercoagulable, normal or hypocoagulable. The coagulation activator is added in an amount that triggers a thrombin explosion that is dependent on the propagation phase and amplification pathways. In this way, a single assay can assess the hemostatic potential of a sample.

FIELD OF THE INVENTION

[0001] The present invention is related to U.S. Pat. No. 5,646,046 toFischer et al and U.S. Pat. No. 6,101,449 to Givens et al, the subjectmatter of each being incorporated herein by reference. The invention isdirected to a method for determining whether a patient ishypercoagulable, hypocoagulable or normal in a single test on a samplefrom the patient. The invention allows for globally assessing both thehypercoagulable potential and hypocoagulable potential of a patient in asingle assay.

BACKGROUND OF THE INVENTION

[0002] Hemostasis is the entire physiological process of maintainingblood in a fluid state within intact blood vessels and preventing excessblood loss by arresting flow via the formation of a hemostatic plug.Normal hemostasis is maintained by tightly regulated interactions of theblood vessel wall, blood platelets and blood plasma proteins. Undernormal conditions there is a delicate balance between the individualcomponents of the hemostatic system. Any disturbances in this hemostaticbalance, the hemostatic potential, could result in bleeding orthrombosis, FIG. 1. By “hemostatic potential” we mean the ability tomaintain a balance between procoagulant and anticoagulant states, asmeasured by fibrin polymerization, when coagulation is initiated by atrigger or activator.

[0003] A thrombotic tendency (thrombophilia) results from the generationof excess thrombin activity and increased fibrin polymerization and clotformation (hypercoagulability) while a bleeding tendency (hemophilia)results from insufficient thrombin generation and reduced fibrinpolymerization and clot formation (hypocoagulability). There is as yetno single laboratory parameter that is increased in all forms ofhypercoagulability and decreased in all forms of hypocoagulability. Thisis in part due to factors other than plasma that play a part inhemostasis. As described above, these other factors include the bloodvessel wall and platelets. However, large proportions of the hemostaticdisorders are related to defects or deficiencies in the blood proteinsthat constitute the coagulation system. These proteins are responsiblefor the stabilization of the platelet plug by the formation of fibrin.Therefore, a global measure of the plasma contribution to coagulationwould facilitate the investigation and management of patients withaltered hemostasis.

[0004] Thrombophilia and haemophilia can be either congenital oracquired. The congenital forms have a genetic basis and are thereforenot readily corrected. The acquired forms generally result fromenvironmental changes, often the effect of drugs, and are thereforesusceptible to manipulation. For example a normal individual givenwarfarin develops acquired haemophilia, stopping the warfarin abolishesthe condition. A normal individual given high dose estrogen developsacquired thrombophilia, stopping the estrogen abolishes the condition.The fundamental basis of both the congenital (genetic) and acquired(environmental) thrombophilias and haemophilias is a change in eitherthe amount or activity of one or more key components of the coagulationpathway. For example the most commonly recognized hereditary form ofthrombophilia is a mutation in the factor V gene which results in theproduction of a structurally altered factor V protein (Factor V Leiden)that is resistant to enzymatic cleavage by protein C, a criticalregulatory component. Classical Haemophilia A is due to a mutation inthe factor VIII gene which results in either reduced production offactor VIII, or production of a structurally altered factor VIII proteinthat does not function correctly. In contrast to the congenitalthrombophilias and haemophilias the acquired forms do not result fromaltered structure but rather alteration of the amount of a keycomponent, typically more than one at a time. For example thethrombophilic effect of oestrogen is due to the composite effects of arise in factors XI, IX, VIII, II and fibrinogen and a reduction in theanticoagulant protein S. The haemophilic effect of warfarin is due to areduction in factors II, VII, IX and X. FIG. 2 illustrates the variousstates of coagulability and lists examples of assays used to assess thedegree or presence of an imbalance. There is currently not an assay thatcan be used to assess both hyper and hypocoagulability simultaneously.This is due in part to the complexity of the coagulation process, theinterdependence of the various components and the identification of ameans to monitor the hemostatic potential of the entire coagulationsystem. FIG. 3 presents an overview of the coagulation process. Theprocess can be divided into four dependent phases, (1) the initiationphase, (2) the propagation phase, (3) the amplification phase and (4)the polymerization phase. All of the phases are affected by regulatoryand feedback processes referred to as anticoagulant pathways.

[0005] Initiation or triggering of coagulation occurs by exposure oftissue factor due to vascular damage, plaque rupture or monocyteexpression as a result of inflammation. Trace amounts of FVIIa andtissue factor form the extrinsic Xase complex. This complex enhances thecatalytic activity of Vila towards factors X and IX resulting in theformation of the active enzymes Xa and IXa. Factor Xa generated by theextrinsic Xase complex forms a small amount of thrombin (IIa). Thethrombin generated is capable of activating small amounts of thecofactors VIII and V. In vivo, the extrinsic Xase complex is quicklyinactivated by Tissue Pathway Factor Inhibitor, TFPI, via the formationof a quaternary complex consisting of TF, VIIa and Xa. Underphysiological conditions the extrinsic Xase generates only picomolaramounts of thrombin.

[0006] During the propagation phase of coagulation the role of theextrinsic Xase is minimized and Factor Xa is alternatively generated bythe complex of the enzymes IXa and its cofactor Villa. This enzymecomplex is referred to as intrinsic Xase. Formation of the Xa by theintrinsic Xase complex is approximately 50 fold more efficient than theextrinsic Xase. Factor Xa and its activated cofactor, FVa, form acomplex on the surface of activated platelets. This is an efficientcatalyst for the conversion of prothrombin to thrombin, referred to asthe prothrombinase complex. Thrombin formed via the intrinsic Xasecomplex is capable of amplifying its own production by positive feedback(activation). Thrombin activates Factors VIII and V and Factor XIactivation leads to further production of the enzymatic component ofintrinsic Xase (Factor IXa). Normal thrombin production is highlyregulated and localized. TFPI neutralizes the trigger for thrombingeneration. Active proteases (IIa, Xa, IXa) must be inactivated byprotease inhibitors to avoid disseminated thrombosis. One of the mostsignificant of these inhibitors is antithrombin III (ATIII). Boththrombin and Xa, and to a lesser extent IXa released from membranesurfaces, are rapidly inhibited by ATIII. Thrombin can also bindnon-damaged sub-endothelium via a receptor molecule, Thrombomodulin(TM). The formation of the IIa/TM complex changes the substratespecificity of thrombin from a procoagulant to an anticoagulant.Thrombin bound to TM is a potent activator of Protein C, converting itto the active enzyme Activated Protein C (APC). APC together with itscofactor protein S cleaves activated cofactors FVIIa and FVa yieldingtheir inactive forms, FVIIIi and FVi. Thrombomodulin also acceleratesthe inactivation of thrombin by ATIII.

[0007] The formation of thrombin leads ultimately to cleavage offibrinogen to form fibrin. During the polymerization phase cross-linkingof soluble fibrin strands is mediated by Factor XIIIa, an enzymegenerated by thrombin activation. The thrombin-TM complex activates theprocarboxypeptidase thrombin activated fibrinolysis inhibitor (TAFI).Thus thrombin plays a role during this phase by both influencing thearchitecture and stabilization of the fibrin clot. Thrombin is a keyenzyme and effector of the coagulation process. Thrombin is both apotent procoagulant and anticoagulant. However, it is thrombin's abilityto cleave fibrinogen and its contribution to fibrin polymerizationevents that are critical to maintaining stasis.

[0008] Clot initiation, often referred to as clotting time, occurs atthe intersection between the initiation and propagation phases when onlyapproximately 5% of thrombin has been formed. The majority of thethrombin formed is generated after the initiation of fibrinpolymerization, thus the rate of fibrin polymerization is a moresensitive indicator of the dynamics of coagulation. Changes in thepropagation phase, amplification phase and anticoagulant pathways alterthe rate of thrombin generation and the impact of thrombin availabilityon rate of fibrin polymerization. Recent studies by Cawthern et al.(1998) suggested that measurement of this thrombin is more informativethan clotting time in assessing the pathophysiology of hemophilias.However these investigators measured thrombin by looking at the kineticsof formation of the thrombin-antithrombin complex (indictor of thrombingeneration) and formation of fibrinopeptide A (indicator of fibrinogencleavage) and not by measuring the kinetics of fibrin polymerization.Variations in concentration or quality of the fibrinogen or fibrinstrands can only be measured as a function of the actual polymerizationprocess. Assays currently used to assess variations in the coagulationprocess typically can only assess variations in one or two phases. Theseassays measure events independently and therefore negate or eliminatethe ability to detect variations in the other phases or interactionsbetween the various phases.

[0009] Assays associated with the assessment of bleeding risk includethe Prothrombin Time (PT), Activated Partial Thromboplastin Time (aPTT),Thrombin Time (TT) and Fibrinogen (Fib) assays (FIG. 2). These assaysare based on the addition of potent activators of the coagulationprocess and thus are only abnormal when major defects are present. Theseassays are not designed to detect the composite effect of multiple minoralterations. For example in the PT test, which utilizes a very highconcentration of a tissue extract, called thromboplastin, and calciumare added to citrated plasma. Whole blood is mixed with citrate when theblood sample is taken. The citrate binds the calcium and“anticoagulates” the blood as calcium ions are required for assembly ofthe tenase and prothrombinase complexes. The blood sample is thencentrifuged and the plasma is separated. When calcium is added back, thetenase (or Xase) and prothrombinase complexes can form and thrombin canbe generated. The source of tissue factor is the thromboplastin.However, the concentration of tissue factor is extremely high(supraphysiological) and so only the initiation phase of thrombingeneration is required. The propagation and amplification phases arebypassed. The prothrombin time is therefore insensitive to many changesin the coagulation pathway and is incapable of detectinghypercoaqulability. Assays based on diluted thromboplastin have beenformulated to aid in the diagnosis of patients with antiphospholipidsyndrome (APS). In these methods the thromboplastin together with thephospholipids are diluted to enhance the sensitivity of the PT to thepresence of antiphospholipid antibodies. The dilute PT clotting time isprolonged in APS due the unavailability of phospholipid surfaces andtherefore the assay is phospholipid dependent instead of TF dependent.

[0010] Assays associated with the assessment of a hypercoagulable state(FIG. 2) include the Thrombin Anti-Thrombin Complex (TAT), Prothrombinfragment F1.2, PAI 1, APCr and D-dimer. These assays are designed tomeasure a specific marker or product of the coagulation process. Forexample, the measurement of elevated levels of D-dimer indicates thatthe clotting process has been activated. However, there is no way ofdetermining whether the D-dimer was being produced as a product of thenormal healing process or if there is an underlying hypercoagulablerisk. The hypercoagulable state cannot be globally assessed by a singleassay but currently requires a battery of tests. A global assay for theassessment of hemostatic potential would be able to identify animbalance utilizing a single assay principle that is sensitive todefects, singular or in combination. The assay would also be sensitiveto effects of intervention to restore the hemostatic balance.

[0011] Recognising the limitations of the screening assays available forhypcoagulable assessment and the battery of assays required forhypercoagulable assessment, others have tried to develop global tests.These tests were designed to be sensitive to the amount of thebiological components and their interactions, as well as measure thedynamics of thrombin generation including regulation. The thrombingeneration curve was described more than 30 years ago as a measure ofthe thrombin generating potential of plasma. A modification of thethrombin generation curve has been described with quantification ofthrombin with a exogenously added chromogenic substrate. This has beencalled the endogenous thrombin potential (ETP). The assay assumes thatthere is a direct correlation between endogenous thrombin potentialmeasured via an exogenously added artificial substrate and theassessment of a hemostatic imbalance. The use of an artificial substrateinstead of thrombin's natural substrate, fibrinogen, ignores the effectsof variations in fibrinongen concentration and fibrinogen configuration.Thrombin is a cleavage product from the proteolysis of Prothrombin, aserine protease. Thrombin then cleaves fibrinogen, its naturalsubstrate, resulting in soluble fibrin monomers that are crossed linkedvia FXIIIa to formed crossed linked polymerized clots. Thrombin is ahighly regulated molecule that possesses both procoagulant andantithrombotic behavior. Additionally, there are numerous substratesthat inactivate thrombin before it can cleave fibrinogen. In addition tonot directly measuring the ability to form a clot the ETP assay hasseveral other major limitations. Limitations of the test include:

[0012] 1. The plasma sample must be defibrinated, typically with a snakevenom. Defibrinating snake venoms activate FX and they also cleave thechromogenic substrate used to quantitate thrombin. This can cause avariable over-estimate of the thrombin potential.

[0013] 2 The plasma sample is considerably diluted in order to prolongthe dynamics of thrombin generation. This results in a non-physiologicalregulation of the thrombin explosion.

[0014] 3 The technique involves multiple subsampling at specifiedtimepoints. For example, a computer linked pipeting device designed inorder to terminate thrombin activity in the subsamples exactly at aspecified time. It is possible to perform the assay manually but it isbeyond the ability of many technologists and requires considerableskill. The test cannot be automated on standard clinical laboratorycoagulometers.

[0015] 4 The formation of thrombin-α2 macroglobulin complex leads toover-estimation of the thrombin potential. A complex mathematicalmanipulation of the results to approximate it to the true thrombinpotential is therefore required.

[0016] 5 Does not take into account the rate or ability of thrombin tocleave fibrinogen.

[0017] Duchemin et al. described a further modification of the ETP wherethe protein C pathway is assessed by adding exogenous thrombomodulin.This method was also modified to take into account proteins thatmodulate anticoagulant activity, including antithrombin III. Like ETP,this modified assay is designed to only measure thrombin generation andnot the effects of thrombin, i.e. dynamic clot formation.

[0018] Other investigators have attempted to design assays sensitive tothe composite of biological components of the coagulation process andtheir interactions. One such example is described by Kraus (Canadianapplication 2,252,983). The method is however limited to determining theanticoagulant potential of a sample by adding thrombomodulin andthromboplastin in a coagulation test. In the described method theemphasis is on dilutions of thromboplastin such that thrombin isproduced at a rate slow enough to enable sufficient activation ofprotein C during the measuring time of the coagulation apparatus. Adisadvantage of this method is that because it depends on clot time, theamount of thromboplastin is more restrictive and higher concentrationsare required to compensate for increases in clotting time whenthrombomodulin is added. Because the method described is aimed atassessing anticoagulant potential and not global hemostatic potentialthe assay is not sensitive to defects in the propagation andamplification phases, the kinetics of clot polymerization or to theinterrelationships between the factors responsible for thrombingeneration.

[0019] The present invention however assesses both the anticoagulant andprocoagulant potential of a blood sample. Furthermore, the presentinvention's sensitivity can be enhanced by using more dilute coagulationactivator, more dilute than has previously been used, since the endpointmethod is not restricted to clot time but analysis can be conducted forthe entire dynamic coagulation process as measured by evaluating kineticparameters of the optical data profile. Analysis of more than simplyclot time can be accomplished even when very weak and unstable clots areformed.

[0020] Variations in the amplification and/or propagation phases willreduce or alter the rate of generation of thrombin and thus impact therate of fibrinogen cleavage and ultimately the rate of fibrinpolymerization. Because the present invention can measure the rate offibrin polymerization throughout the dynamic coagulation process, itmeasures the clinically important thrombin that is generated afterclotting time.

[0021] Other prior art (Mann et. al.) assesses coagulation problems bytaking a series of independent and indirect measurements. Thrombingeneration is measured as a function of TAT complex formation or the useof a chromogenic substrate and the formation of fibrin as measured bythe release of FPA. All of the systems and models to date have beendesigned to understand a discrete process or interaction of thecoagulation process and cannot provide an assessment of the overallhemostatic potential. In contrast, the method of the present inventionis designed to not only assess the interplay of the coagulation proteinstogether with synthetic cell surfaces, it is aimed at capturing this ina dynamic measurement that correlates to clinical outcome. Thetechnology and methods described in the present invention can also bemodified to introduce components of the fibrinolytic system as well ascells and flow conditions.

[0022] Givens et. al. demonstrated that a model which characterizes theprocess of clot formation and utilizes parameters in addition toclotting time is sensitive to defects in the clotting proteins. Table 1describes the parameters defined by Givens et al. and FIGS. 4 and 5illustrate how those parameters are determined and how they relate tofibrin polymerization for the PT and aPTT assays. However, this work wasconducted utilizing data from the PT and APTT assays, which as discussedearlier, are only sensitive to events associated with the hypocoagulablestate. Additionally, the work described was conducted in the presence ofstrong clot formation because of the addition of supraphysiologicalconcentrations of tissue factor. Fibrin polymerization is significantlyaltered in a dilute systems designed for global hemostatic assessmentresulting in weak and unstable clot formation. Global hemostaticassessment and new methods for monitoring and quantifying fibrinpolymerization are required.

SUMMARY OF THE INVENTION

[0023] In order to overcome the deficiencies in the prior art as notedabove, a global test of coagulation has now been developed, which isaccurate and easy to use. With the present invention, a single test canbe used to quantify both hyper- and hypocoagulability. The concept isbased on the addition of a minimal concentration of coagulationactivator sufficient to trigger but insufficient to result in completefibrin polymerization so as to allow detection of perturbances in thepropagation, amplification and polymerization pathways. In a dilutesystem, the coagulability (hyper/hypo) of a sample determines themagnitude of the thrombin explosion and the direct and indirectinfluence that has on the rate of fibrin polymerization. This concept iscontrary to an assay system such as the PT, which uses excess amounts ofTF (or thromboplastin). In the method of the present invention,therefore, disturbances in the propagation and amplification loops areaccessible, whereas in the traditional PT test, these parts of thecoagulation pathway are overshadowed by the excessive amounts of FactorIIa produced by the initiation phase.

[0024] In one embodiment of the invention, the rate of fibrinpolymerization produced by a standardized coagulation activator dilutionis then used to indicate if a plasma sample is normal, hyper- orhypocoagulable. In addition, the technique can be used to determine howmuch the plasma needs to be modified in order to restore coagulabilityto normal. For example, in the case of hypocoagulability, this might beachieved by clotting factor replacement or in the case ofhypercoagulability, by the addition of a natural anticoagulant or theuse of an anticoagulant drug.

[0025] In the present invention, at a given coagulation activatordilution, the rate of fibrin polymerization of haemophilia plasmas areless than the rate of polymerization for a normal plasma and the rate offibrin polymerization of thrombophilia plasmas are greater than that ofa normal sample. The rate of fibrin polymerization is sensitive to minorchanges in the components of hemostasis even when differences inclotting time cannot be detected. FIG. 6 illustrates waveforms from thenormal, hypercoagulable and hypocoagulable specimens. The rate ofpolymerization is affected even though the time of clot initiation isessentially unchanged.

[0026] In another embodiment of the present invention, a test isprovided that can be used to determine the degree of hyper- orhypocoagulability of a plasma sample. Furthermore, it can be used onsamples containing platelets or other cells as a measure of thecontribution of cellular components to coagulability. The test, in someembodiments, relies on the use of a standardized dilution ofthromboplastin in the presence of an excess of phospholipids with therate of fibrin formation as the detection endpoint. The test is simpleand can be automated on standard laboratory coagulometers. The test inthe present invention can be run on a test sample in the absence of theaddition of an exogenous substrate, e.g. a chromogenic substrate. Thetest is sensitive to fibrin concentration and/or configuration.

[0027] In a further embodiment of the invention modifications to thecomponents or concentrations of the reagent or endpoint selection aretailored to facilitate the development and/or monitoring of novelpharmaceutical agents. Examples of such applications are inhibitors ofinitiation of the TF pathway (TFPI, FVIIa inhibitors), inhibitors ofthrombin generation such as inhibitors of FXa, (syntheticpentasaccharides) and inhibitors of thrombin activity (direct thrombininhibitors). Lipid composition, size or concentration can also bemodified to tailor the assay towards the development of drugs targetedto the propagation and amplification pathways. For example, lipidcomposition can be altered to produce vesicles that maximize Xageneration or alternatively, designed to maximize prothrombinaseactivity. Thus the efficacy of inhibitors of Xa and those directed atthe prothrombinase complex may be assessed. The invention can also bemodified to focus on the anticoagulant potential of the plasma byincluding thrombomodulin, an activator of protein C. Lipid vesiclesmaximizing the activity of APC could also be added to the reagent. Theassay can also be modified to exaggerate a mildly abnormalsubpopulation. The consequences of this approach are that severelythrombotic or hemorrhagic samples will exceed the signal to noise ratioand not be measured but subtle differences at the onset of a disease oran earlier indication of effective intervention would be gained.Endpoint selection and ratios derived from comparison to known sampleswould be exploited to further improve sensitivity and specificity of thereagent modifications. These approaches would therefore be utilized inthe drug discovery and drug development processes where assay designedfor a global assessment of the hemostatic potential are required.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 illustrates the consequences of any disturbance in thisso-called hemostatic balance or potential.

[0029]FIG. 2 illustrates the conditions associated with being out ofhemostasis and lists examples of assays used to assess the degree orpresence of an imbalance.

[0030]FIG. 3 illustrates the four dependent phases of the coagulationprocess.

[0031]FIG. 4 illustrates the optical data from a clotting assay and thefirst and second derivative calculated from that data.

[0032]FIG. 5 illustrates where min_(—)2, the time index of min2(clotting time), min_(—)1, max_(—)2 and delta (proportional tofibrinogen concentration) are located in the optical data profile.

[0033]FIG. 6 illustrates examples of waveforms for the global screeningassay at dilute tissue factor.

[0034]FIG. 7 illustrates the change in ratio as a function of dilutionfor a FVIII deficient specimen and a Protein S deficient Specimen.

[0035]FIG. 8 illustrates ratios of the min-1 values (the maximum rate offibrin polymerization) for hypocoagulable specimens at three dilutionsof rTF compared to the min_(—)1 values of the ratio of the same dilutionof a normal plasma.

[0036]FIG. 9 illustrates ratios of the min_(—)1 values forhypercoagulable specimens at three dilutions of rTF and 10 nMthrombomodulin compared to min_(—)1 values of the ratio for the sameconditions of a normal plasma.

[0037]FIG. 10 illustrates the effects on min_(—)1 values of varyingtissue factor and thrombomodulin concentrations on results forhypercoagulable, hypocoagulable and normal plasmas.

[0038] Abbreviations in the figures are as follows:

[0039] Activated Factor IX (FIXa)

[0040] Activated Factor V (FVva)

[0041] Activated Factor VII (FVIIa)

[0042] Activated Factor VIII (FVIIa)

[0043] Activated Factor X (FXa)

[0044] Activated Factor XI (FXIa)

[0045] Activated Factor XIII (FXIIIa)

[0046] Activated Protein C (APC)

[0047] Factor II (FII)

[0048] Factor IX (FIX or F9)

[0049] Factor V (FV)

[0050] Factor V Leiden (FVL)

[0051] Factor VII (FVII)

[0052] Factor VIII (FVIII or F8)

[0053] Factor VIII Deficient (FVIII-def)

[0054] Factor X (FX)

[0055] Factor XI (FXI)

[0056] Factor XIII (FXIII)

[0057] George King (GK)

[0058] HRF (Hemophilia Research Foundation)

[0059] Organon Teknika Normal Pool Plasma (OT NPP)

[0060] Protein C (PC)

[0061] Protein C Deficient (PC Def.)

[0062] Protein S (PS)

[0063] Protein S Deficient (PS-Def)

[0064] Prothrombin Mutation 20210 (PT 20210)

[0065] Recombinant Tissue Factor (rTF)

[0066] Thrombin or aActivated Factor II (FIIa)

[0067] Thrombomodulin (TM)

[0068] Tissue Factor (TF)

[0069] Von Willebrand Factor (vWF)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0070] The present invention is directed to a method for determining ifa patient or specimen from said patent is hypercoagulable,hypocoagulable or normal in a single test, and comprises the steps ofinitiating coagulation in a patient's sample in vitro in the presence ofan activator. Said activator is added to the sample in an amount whichwill result in intrinsic tenase-dependent fibrin polymerization(involves propagation and amplification loops). Preferably the plasmasample is undiluted thus allowing for sufficient concentrations of allof the endogenous proteases and inhibitors. Formation of the fibrinpolymerization is recorded over time so as to derive a graphictime-dependent polymerization profile. This profile will show whetherthe patient is hypercoagulable, normal, or hypocoagulable by comparingthe sample profile with a profile from a known sample.

[0071] Preferably, the activator is a thromboplastin, more preferablyTissue Factor (TF). In its most preferred form, the TF is recombinant TF(rTF) that is relipidated with phospholipids, which form liposomevesicles. Preferably phospholipids provide the surfaces to assembleintrinsic Xase and prothrombinase complexes. The phospholipids arepresent at a concentration, which is not rate limiting to thecoagulation process and remains constant and independent of dilution.These phospholipid vesicles mimic platelet and monocyte surfaces.

[0072] Optical data profiles are generated on an automated coagulationanalyzer such as the MDA™ 180 offered by Organon Teknika Corporation.Preferably endpoints such as the time of clot initiation and the rate ofpolymerization are calculated from the data profiles. More preferablythe 1^(st) and 2^(nd) derivatives from the data profile are calculatedand the min and max of the derivatives are calculated with respect tovalue and the associated time index. Most preferably the endpoints arecalculated and one or more of the following ratios are calculated usingthe mentioned endpoints:

[0073] Option 1—Endpoint(s)

[0074] Option 2—Ratio at different dilutions (ratio 1)

[0075] Endpoint (z) for dilution (x)

[0076] Endpoint (z) for dilution (y)

[0077] Option 3—Ratio of dilutions compared to normal (ratio 2)

[0078] Ratio 1 for patient sample

[0079] Ratio 1 for normal plasma

[0080] Option 4—Ratio for different reagent formulations

[0081] Ratio 2 with formulation (a)

[0082] Ratio 2 with formulation (b)

[0083] Option 5—Ratio of different endpoints

[0084] Ratio 2 with endpoint (z)

[0085] Ratio 2 with endpoint (z′)

[0086] Option 6—Ratio of specimen to normal at a given dilution

[0087] Endpoint (z) at dilution x for a specimen

[0088] Endpoint (z) at dilution x for a normal plasma

[0089] Additionally, other ratios, differences or models to normalizethe assay can be calculated. The normal plasma can be substituted withany known plasma. Known plasma is defined as a plasma that has beencharacterized with respect to a condition of the specimen.

[0090]FIG. 1 illustrates the consequences of any disturbance in thisso-called hemostatic balance or potential. Too little hemostasis(decreased platelet function, hypo-coagulation, hyper-fibrinolysis) atthe site of injury leads to persistent bleeding, while too muchhemostasis (increased platelet function, hyper-coagulation,hypo-fibrinolysis) leads to the formation of an excessive thrombus withvascular obstruction and ischemia.

[0091]FIG. 2 illustrates the conditions associated with being out ofhemostasis and lists examples of assays used to assess the degree orpresence of an imbalance.

[0092]FIG. 3 illustrates the four dependent phases of the coagulationprocess, (1) the initiation phase, (2) the amplification phase, (3) thepropagation phase and (4) the polymerization phase of hemostasis. All ofthe phases are affected by regulation and feedback processes referred toas anticoagulant pathways.

[0093]FIG. 4 illustrates the optical data from a clotting assay and thefirst and second derivative calculated from that data. Table 1 describesa set of parameters calculated from the data and derivatives illustratedin FIG. 4. TABLE 1 Parameter Description Slope 1 Initial slope frompoint A to point B Delta 1 Amplitude of signal change from point A topoint B Slope 3 Final slope from point D to point E Delta Amplitude ofsignal change Index Min 1 Time at point C Min 1 Minimum value of 1stderivative (Rate of change at point C) Index Max 2 Time at point D Max 2Max. value of 2nd derivative (Acceleration at point D) Index Min 2 Timeat point B Min 2 Minimum value of 2nd derivative

[0094]FIG. 5 illustrates where min_(—)2, the time index of min2(clotting time), min_(—)1, max_(—)2 and delta (proportional tofibrinogen concentration) are located in the optical data profile.

[0095]FIG. 6 contains examples of waveforms for the global screeningassay at dilute tissue factor. The APC resistant, , hypercoagulablespecimen, generates a waveform that has essentially the same time ofclot initiation compared to the normal. However, the rate of fibrinpolymerization for the hypercoagulable specimen is significantly greaterthan that of the normal. The FVIII and FIX deficient hypocoagulablespecimens, have only a slightly prolonged time of clot initiationwhereas the rates of polymerization are significantly reduced whencompared to normal or hypercoagulable specimens.

[0096]FIG. 7 illustrates the change in ratio as a function of dilutionfor a FVIII deficient specimen and a Protein S deficient Specimen. Theratio values at 1:50,000 dilution of thromboplastin deviate from theresponse of the normal plasma. The hypocoagulabe specimen producesratios that are greater than 1 and the hypercoagulable specimen hasratios that are less than 1 for this endpoint (clot time)/ratiocombination. Additionally, the abnormal specimen deviates from normal atdifferent dilutions and in opposite directions.

[0097]FIG. 8 contains ratios of the min-1 values (the maximum rate offibrin polymerization) for hypocoagulable specimens at three dilutionsof rTF compared to the min_(—)1 values of the ratio of the same dilutionof a normal plasma. All of the ratios of the hypocoagulable plasmas forall three dilutions are less than the normal response (values of <1). Asthe dilution increase, i.e. less tissue factor is provided, thedifference in the ratios increases.

[0098]FIG. 9 illustrates ratios of the min_(—)1 values forhypercoagulable specimens at three dilutions of rTF and 10 nMthrombomodulin compared to min_(—)1 values of the ratio for the sameconditions of a normal plasma. All of the ratios of the hypercoagulableplasmas for all three dilutions are greater than the normal response(values of >1). As the dilution increase, i.e. less tissue factor isprovided, the difference in the ratios increases.

[0099]FIG. 10 illustrates the effects on min_(—)1 values of varyingtissue factor and thrombomodulin concentrations on results forhypercoagulable, hypcoagulable and normal plasmas. The data indicatethat an optimal concentration can be defined to facilitatedifferentiation between normal, hypercoagulable and hypocoagulableplasmas. Additionally, other concentrations of tissue factor andthrombomodulin facilitate improvements in sensitivity and specificityfor a particular condition at the expense of the sensitivity andspecificity of another type of condition.

[0100] Tables 2 and 3 summarize the results of measuring the kineticparameters, min 1 and min 2 with a series of defined patient plasmas.The concentration of TF was 10 μM and TM was adjusted to 10 nM. Thephospholipid concentration was kept constant at 150 micromolar. The datashows that the reagent in the presence of TM is able to differentiatehyper and hypocoagulable plasmas with a single reagent formulation.Additionally, the data indicates that TM is not essential to obtaindiscrimination between the hypocoagulable and a normal standard plasmapool. Data are calculated as ratios to a normal pool with and withoutthrombomodulin. Ratios of the min2 parameter were higher than thecorresponding min1 values for the hypercoagulable plasmas.

[0101] Tables 2 and 3 illustrate the behavior of defined plasmas in thepresence and absence of thrombomodulin as determined by the kineticendpoints min_(—)1 and min_(—)2. TABLE 2 Min_1 Ratio Min_1 RatioSpecimen without Specimen with 10 nM Min_1 values with no Min_1 valueswith 10 TM/Normal plasma TM/Normal plasma Plasma Type TM nM TM withoutTM with 10 nM TM Normal Plasma 101 68 PC Deficient 110 105 1.09 1.54Lupus 116 79 1.15 1.16 FV Leiden 95 77 0.94 1.13 FV Leiden & 260 2482.57 3.64 PT 20210 FIX Deficient 71 40 0.70 0.59 FVIII Deficient 84 460.83 0.68

[0102] TABLE 3 Min_2 Ratio Min_2 Ratio Specimen without Specimen with 10nM Min_2 values with no Min_2 values with 10 TM/Normal plasma TM/Normalplasma Plasma Type TM nM TM without TM with 10 nM TM Normal Plasma 34.811.9 PC Deficient 36.4 27.6 1.05 2.32 Lupus 47.4 23.6 1.36 1.98 FVLeiden 32.6 20.3 0.94 1.71 FV Leiden & 181 165 5.2 13.9 PT 20210 FIXDeficient 21 9 0.60 0.76 FVIII Deficient 16.4 6.4 0.47 0.54

EXAMPLE 1

[0103] The assay was conducted by adding 50 uL of plasma to 50 uL of theactivator and then adding 50 uL of the start reagent. A normal sample, ahypocoagulable sample (Factor VIII deficient plasma) and ahypercoagulable plasma (protein S deficient plasma) were evaluated atvarious dilutions of the activator. The activator was a commerciallyavailable thromboplastin (Thromborel R, Behring Diagnostics) dilutedwith a buffer at two dilutions, a 1:100 and 1:50000 of its originalconcentration. The start reagent consisted of 0.25 M Calcium Chloride.The assay was conducted at 37 C and the reaction was monitored at 580 nmfor 300 seconds. Endpoints were calculated for time and rate indices ofclot formation. Ratios of the endpoints were compared to other dilutionsand other samples as follows:${Ratio} = \frac{{{endpoint}\quad {of}\quad {reagent}\quad {diln}\quad (x)\quad {for}\quad {{Specimen}/{endpoint}}\quad {of}\quad {reagent}\quad {{diln}{\quad \quad}(y)}\quad {for}\quad {specimen}}\quad}{{{endpoint}\quad {of}\quad {reagent}\quad {diln}\quad (x)\quad {for}\quad {{npp}/{endpoint}}\quad {of}\quad {reagent}\quad {diln}\quad (y)\quad {for}\quad {npp}}\quad}$

[0104] Where x is a 1:100 dilution and y is a series of dilutions

[0105] As the dilution of the reagent become greater (y becomes larger)the results for the two abnormal plasmas (the aforementionedhypercoagulabe and hypocoagulable plasmas) tested began to deviate fromthe calculated endpoints or ratios of the normal plasma. The results canbe expressed as the magnitude of deviation at a given dilution or as thedilution required to deviate from ideal (normal value or normal range).FIG. 7 illustrates that the hypercoagulable and hypocoagulable resultsdeviate in opposite directions indicating the ability to differentiatebetween the two conditions.

EXAMPLE II

[0106] The assay was conducted by adding 50 uL of plasma to 50 uL of theactivator and then adding 50 uL of the start reagent. A set of normalsamples, a series of samples from hypocoagulable individuals and aseries of plasmas from hypercoagulable individuals were evaluated atvarious dilutions of the activator. The activator was a preparation ofTF reconstituted with phospholipids to between 20 to 3.3 pM (1:20,000 to1:120,000 dilution) and phospholipid prepared by extrusion with andwithout TM. The start reagent consisted of 0.025 M Calcium Chloride. Theassay was conducted at 37 C and the reaction was monitored at 580 nm for300 seconds. The value of the minimum of the 1^(st) derivative and thevalue of the minimum of the 2^(nd) derivative were calculated for allsamples. Ratios of the endpoints were compared to other dilutions andother samples as follows:

[0107] Option 1—Endpoint(s)

[0108] Option 2—Ratio at different dilutions (ratio 1)

[0109] Endpoint (z) for dilution (x)

[0110] Endpoint (z) for dilution (y)

[0111] Option 3—Ratio of dilutions compared to normal (ratio 2)

[0112] Ratio 1 for patient sample

[0113] Ratio 1 for normal plasma

[0114] Option 4—Ratio for different reagent formulations

[0115] Ratio 2 with formulation (a)

[0116] Ratio 2 with formulation (b)

[0117] Option 5—Ratio of specimen to normal at a given dilution

[0118] Endpoint (z) at dilution x for a specimen

[0119] Endpoint (z) at dilution x for a normal plasma

[0120]FIGS. 8 and 9 illustrate the differentiation for hypercoagulableand hypercoagulable specimens when compared to normal. Tables 2 and 3illustrate the behavior of defined plasmas in the presence and absenceof thrombomodulin as determined by the kinetic endpoints min_(—)1 andmin_(—)2. FIG. 10 demonstrates the effect of varying tissue factor andthrombomodulin on the results from hypercoagulable, hypercoagulable andnormal plasmas. The data indicate that variations in the concentrationsfacilitate improvements in sensitivity and specificity for a conditionat the expense of the sensitivity and specificity of another type ofcondition.

[0121] In one preferred embodiment, the TF is added to the sample at aconcentration of about less than or equal to 10 picomolar and thephospholipid concentration of between 10 to 300 μM. The TF can be addedto the sample at a concentration of 3 to 10 picomolar and thephospholipid vesicles can be added at 100 to 150 micromolar. Preferablythrombomodulin is added at 0 to 30 nanomolar and most preferably at aconcentration of 5 to 15 nanomolar. Calcium Chloride is most preferablyadded at a concentration of about 25 mM. All of the reagent componentconcentrations described are further diluted 1:3 in the plasma/buffermatrix in the cuvette.

[0122] One or more parts or endpoints of the time dependent measurementprofile obtained by monitoring fibrin polymerization in the test samplecan be compared to the same parts or endpoints of a time dependentmeasurement profile obtained by monitoring fibrin polymerization in thetest sample at a different coagulation activator concentration and/or tothe same parts or endpoints for a known (e.g. normal) test sample. Thepart of the profile can be one or more of initiation of clot formation,overall change in profile, slope of profile after initiation of clotformation, and acceleration at the time of clot initiation. Also, if atleast two time-dependent fibrin polymerization profiles are obtained, anadditional profile can be obtained for a known sample from computermemory or by adding the activator at at least one concentration to aknown sample and monitoring the formation of fibrin polymerization overtime. The parameter from each time-dependent fibrin polymerizationprofile having varying activator concentrations can be determined and aconcentration at which the at least one parameter of said sample beingtested deviates from normal can be determined. The point of deviation isindicative of the hypercoagulable or hypocoagulable state. The part ofthe profile is preferably a time index of the minimum of the firstderivative, the value of the minimum of the first derivative, the timeindex for the minimum of the second derivative, the value for theminimum of the second derivative, the time index of the maximum of thesecond derivative, the value of the maximum of the second derivative, orthe overall magnitude of change. More preferably, the part is rate oracceleration of fibrin polymerization, wherein the rate or accelerationis compared to rate or acceleration at the same activator concentrationfor the known sample.

[0123] Though endpoints can be directly compared as noted above, adifference or ratio of said parameters for said test sample and saidnormal sample can instead be determined. If the parameter is clot time,a ratio of clot times at different activator concentrations can bedetermined. A ratio of other parameters, rate of clot formation, maximumacceleration of clot formation, turbidity at a predetermined timeperiod, and total change in turbidity can also be determined in order tomeasure defects in the thrombin propagation and/or amplification phases.Also, a ratio can be taken of the at least one parameter for said testsample to the same parameter for a normal sample. And, the ratio can bedetermined for multiple concentrations of activator to bettercharacterize the hypo- or hyper-coagulability. For example, theconcentration at which said ratio (test sample/known sample) departsfrom 1 (or a range around 1) can show the abnormal coagulability.

[0124] Other ratios aid determination of the hemostatic potential (e.g.the hypocoagulability, stasis, or hypercoagulability; or the bleeding orthrombotic tendency of the patient). For example, a first ratio can becalculated for the at least one parameter at two differentconcentrations of the activator. A second ratio can be calculated ofsaid first ratio at the two different activator concentrations relativeto a first ratio calculated for a known sample at two differentactivator concentrations. A third ratio can be calculated of the secondratio at a first reagent formulation and the second ratio at a secondreagent formulation. Though the second reagent can vary in a number ofways from the first, in one embodiment the first reagent formulation cancomprise a coagulation activator and the second reagent formulation cancomprise a coagulation activator and an activator of an anticoagulantpathway. A fourth ratio could be calculated of the second ratiocalculated for one endpoint relative to the second ratio calculated fora different endpoint. Significant information can be obtained bychanging the reagent formulation and comparing the same endpoints, or bymaintaining the reagent formulation (though possibly at a differentconcentration) and comparing different endpoints (or both endpoint andreagent formulation and/or concentration can be altered).

[0125] An activator of one or more anticoagulant pathways can be addedalong with the coagulation activator. Such an additional activator canbe any activator of an anticoagulant pathway, such as the protein Cpathway. Thrombomodulin is one example, which can be added in the formof purified human thrombomodulin, purified non-human mammalianthrombomodulin, soluble or membrane associated thrombomodulin, nativethrombomodulin or reconstituted with phospholipids, partially or fullyglycolsylated thrombomodulin, or fully deglycosylated thrombomodulin,with added heparin-like molecules. The coagulation activator can be anysuitable activator including recombinant or purified tissue factor,truncated tissue factor, or cells expressing tissue factor on theirsurface. If vesicles or liposomes are added, they can be in the form ofplatelets, cellular debris, phospholipids or platelet microparticles. Ametal salt if added can be a halide of magnesium, calcium or manganese,or other divalent metal salt. Buffers and stabilizers could also beadded if desired.

[0126] A reagent or kit for assessing hemostatic potential should have acoagulation activator. Additional components of the reagent or kit couldinclude the above-mentioned vesicles, metal salt or ions, andanticoagulant pathway activator, if desired. In the kit, the componentscould all be provided in separate containers, or mixed together in anycombinations in one or more containers. If phospholipid vesicles areadded, they can be any suitable phospholipid or combination ofphospholipids including one or more of phosphatidylcholine,phosphatidylethanolamine and phosphatidylserine, which can be providedat a ratio of approximately 5 to 30 mole percentphosphatidylethanolamine, 1 to 10 percent phosphatidylserine and theremainder phosphatidylcholine. These vesicles can be prepared in avariety of ways to yield liposomes of various sizes. Phospholipids canbe provided at a concentration that is not rate limiting, e.g. at aconcentration of from 10 to 300 micromolar, and preferably in the rangeof from 50 to 200 micromolar. Tissue factor can be provided at aconcentration of 10 picomolar or less, 8 picomolar or less, orpreferably 6 picomolar or less. The concentration could be 3 picomolaror less, though whatever concentration of tissue factor, it should allowfor hemostatic potential assessment as set forth herein. If it isdesired to add thrombomodulin, it can be provided at a concentration of30 nanomolars or less, preferably in a range of from 5 to 20 nanomolar.If a metal salt is to be added, it can be provided in a reagent or kitat a concentration of from 5 to 50 mM, preferably from 15 to 35 mM.

[0127] Variations to the above described method, kit and reagent arepossible, and the embodiments disclosed herein should be consideredillustrative and not limiting.

We claim:
 1. A method for determining if a patient is hypercoagulable,hypocoagulable or normal, comprising: a) providing a test sample fromthe patient; b) initiating coagulation in the sample in the presence ofan activator, which is added to the sample in an amount which willresult in intrinsic tenase-dependent fibrin polymerization; c)monitoring formation of said intrinsic tenase-dependent fibrinpolymerization over time so as to derive a time-dependent profile,wherein results of said fibrin polymerization monitoring determinewhether said patient is hypercoagulable, normal or hypocoagulable. 2.The method according to claim 1, wherein all or part of saidtime-dependent profile is compared to all or part of a time-dependentprofile for a known sample.
 3. The method according to claim 2, whereinpart of said profile is compared, said part of said profile includingone or more of initiation of clot formation, overall change in profile,slope of profile after initiation of clot formation, and acceleration atthe time of clot initiation.
 4. The method according to claim 2, whereinat least two time-dependent fibrin polymerization profiles are obtained,an additional profile being obtained for a known sample from computermemory or by adding said activator at at least one concentration to aknown sample and monitoring the formation of fibrin polymerization overtime.
 5. The method according to claim 4, wherein at least twotime-dependent fibrin polymerization profiles are obtained, one profilefor said test sample at a first activator concentration, and at leastone additional profile for said test sample at a second activatorconcentration and/or one or more profiles for a known sample at one ormore activator concentrations.
 6. The method according to claim 1,wherein the activator comprises tissue factor.
 7. The method accordingto claim 4, wherein at least one parameter from each time-dependentfibrin polymerization profile having varying activator concentrations isdetermined and a concentration at which the at least one parameter ofsaid sample being tested deviates from normal is determined.
 8. Themethod according to claim 7, wherein said at least one parameter isselected from time index and value of the minimum of the firstderivative, the time index and value for the minimum and maximum of thesecond derivative and the overall magnitude of change.
 9. The methodaccording to claim 5, wherein part of each fibrin polymerization profileis compared to a same part of a profile for a known sample.
 10. Themethod according to claim 9, wherein said part is one or more of a timeindex of the minimum of the first derivative, the value of the minimumof the first derivative, the time index for the minimum of the secondderivative, the value for the minimum of the second derivative, the timeindex of the maximum of the second derivative, the value of the maximumof the second derivative, and the overall magnitude of change.
 11. Themethod according to claim 9, wherein said part is rate or accelerationof fibrin polymerization, wherein said rate or acceleration is comparedto rate or acceleration at the same activator concentration for saidknown sample.
 12. The method according to claim 9, wherein a differenceor ratio of said parameters for said test sample and said normal sampleare determined.
 13. The method according to claim 12, wherein saidparameter is clot time and a ratio of clot times at different activatorconcentrations is determined.
 14. The method according to claim 1,wherein one or more parameters of said time-dependent fibrinpolymerization profile are compared to the same one or more parametersfor a normal sample, in order to determine whether said patient ishypercoagulable, normal or hypocoagulable.
 15. The method according toclaim 7, wherein said at least one parameter includes at least one oftime of initiation of clot formation, rate of clot formation, maximumacceleration of clot formation, turbidity at a predetermined timeperiod, and total change in turbidity.
 16. The method according to claim15 wherein said one or more parameters are measures of defects in thethrombin propagation and/or amplification phases.
 17. The methodaccording to claim 15, wherein a ratio of said at least one parameterfor said test sample to the same parameter for a normal sample isdetermined.
 18. The method according to claim 17, wherein said ratio isdetermined for multiple concentrations of activator.
 19. The methodaccording to claim 18, wherein a concentration at which said ratiodeparts from 1 is determined.
 20. The method according to claim 1,wherein an activator of one or more anticoagulant pathways is added. 21.The method according to claim 20, wherein an activator of protein C isadded.
 22. The method according to claim 21, wherein the protein Cactivator is thrombomodulin.
 23. The method according to claim 22,wherein a fibrin polymerization profile is obtained with and withoutsaid thrombomodulin.
 24. The method according to claim 1, whereinmultiple concentrations of said activator are used for providingcorresponding multiple time-dependent measurement profiles, and multipleconcentrations of activator of a known sample are used for providingcorresponding multiple time-dependent known sample measurement profiles,and ratios of one or more parameters of the measurement profiles of theknown and test sample are compared.
 25. The method according to claim24, wherein the one or more parameters at the one or more concentrationsof said activator can be compared in the presence or absence of amodulator of one or more anticoagulant pathways.
 26. The methodaccording to claim 1, wherein one or more parameters at multipleconcentrations of said activator are determined and results arecompared.
 27. The method according to claim 24, wherein anyconcentration of said activator can be compared in the presence orabsence of a modulator of one or more anticoagulant pathways.
 28. Themethod according to claim 27, wherein the activator is tissue factor andthe modulator is thrombomodulin.
 29. The method according to claim 1,wherein the activator comprises tissue factor and phospholipids.
 30. Themethod according to claim 1, wherein a metal salt is added as part ofthe activator or separately therefrom, which metal salt dissociates intoa metal divalent cation when added to the test sample.
 31. The methodaccording to claim 30, wherein the divalent metal cation is magnesium,calcium or manganese.
 32. The method of claim 30, wherein the metal saltis a halide of magnesium, calcium or manganese.
 33. The method of claim1, wherein the activator comprises purified or recombinant tissuefactor.
 34. The method of claim 33, wherein the activator compriseshomogenized cerebral tissue.
 35. The method of claim 1, furthercomprising adding phopholipids together with or separately from theactivator.
 36. The method of claim 1, further comprising adding buffersand/or stabilizers to the test sample.
 37. The method of claim 1,wherein the test sample is a patient plasma sample.
 38. The method ofclaim 2, wherein the known sample is a normal sample.
 39. The method ofclaim 1, wherein the time dependent measurement profile is an opticalabsorbance or transmittance profile provided on an automated analyzer.40. The method of claim 39, wherein a light beam having a wavelength inthe visible spectrum is directed through a container holding the testsample and activator, and light absorbed or transmitted is monitored toform the time dependent measurement profile.
 41. The method of claim 1,wherein the activator comprises tissue factor sufficiently diluted so asto allow determination of any of hypercoagulable, normal orhypcoagulable depending upon the condition of the patient.
 42. Themethod of claim 1, wherein a part of the time dependent measurementprofile other than clot time is compared to the same part of a timedependent measurement profile for a known sample.
 43. The method ofclaim 1, wherein defects in formation of intrinsic tenase complex aredetected.
 44. The method of claim 1, wherein one or more endpoints fromthe time-dependent measurement profile are calculated, the endpointsselected from the time of clot initiation and the rate ofpolymerization.
 45. The method of claim 44, wherein at least oneparameter selected from the first derivative of the time dependentmeasurement profile, the second derivative of the time dependentmeasurement profile, the minimum of the first and/or second derivative,or the maximum of the first and/or second derivative are calculated withrespect to value and/or the time associated time index.
 46. The methodof claim 45, wherein the at least one parameter is compared to the sameat least one parameter for a known sample.
 47. The method of claim 45,wherein a first ratio is calculated for the at least one parameter attwo different concentrations of the activator.
 48. The method of claim47, wherein a second ratio is calculated of said first ratio at the twodifferent activator concentrations relative to a first ratio calculatedfor a known sample at two different activator concentrations.
 49. Themethod of claim 48, wherein a third ratio is calculated of said secondratio at a first reagent formulation and said second ratio at a secondreagent formulation.
 50. The method of claim 49, wherein the firstreagent formulation comprises a coagulation activator and the secondreagent formulation comprises a coagulation activator and an activatorof an anticoagulant pathway.
 51. The method of claim 50, wherein thefirst reagent comprises tissue factor and the second reagent comprisestissue factor and thrombomodulin.
 52. The method of claim 48, wherein afourth ratio is calculated of said second ratio calculated for oneendpoint relative to said second ratio calculated for a differentendpoint.
 53. The method of claim 52, wherein one of the endpoints isclot time and the other is the minimum of the first derivative.
 54. Themethod of claim 1, wherein sample is whole blood or platelet richplasma.
 55. The method of claim 1, further comprising adding vesicles tothe test sample.
 56. The method of claim 55, wherein the vesiclescomprise platelets, cellular debris phospholipid vesicles or plateletmicroparticles.
 57. The method of claim 1, further comprising adding aprotein C activator to the test sample.
 58. The method according toclaim 57, wherein the protein C activator is purified humanthrombomodulin, purified non-human mammalian thrombomodulin, soluble ormembrane associated thrombomodulin, native thrombomodulin orthrombomodulin reconstituted with phospholipids, partially or fullyglycolsylated thrombomodulin or fully deglycosylated thrombomodulin. 59.The method of claim 1, wherein the activator comprises recombinant orpurified tissue factor, truncated tissue factor, or cells expressingtissue factor on their surface.
 60. A method for assessing thecoagulation system in a test sample, comprising: providing a sample tobe tested; adding an activator to said sample to trigger a thrombinexplosion dependent on propagation phase and amplification loops andsubject to one or more anticoagulant pathways; measuring thepolymerization of fibrin due to said thrombin explosion; and assessingthe coagulation system in said test sample based on said measured fibrinpolymerization.
 61. The method of claim 60, further comprising addingvesicles to the test sample.
 62. The method of claim 61, wherein thevesicles comprise platelets, cellular debris, phospholipid vesicles orplatelet microparticles.
 63. The method of claim 60, wherein anactivator of protein C is added to cause the fibrin polymerization to besensitive to the protein C pathway.
 64. The method according to claim63, wherein the protein C activator is purified human thrombomodulin,purified non-human mammalian thrombomodulin, soluble or membraneassociated thrombomodulin, native thrombomodulin or thrombomodulinreconstituted with phospholipids, partially or fully glycolsylatedthrombomodulin or fully deglycosylated thrombomodulin.
 65. The method ofclaim 60, wherein the activator comprises recombinant or purified tissuefactor, truncated tissue factor, or cells expressing tissue factor ontheir surface.
 66. The method of claim 60, wherein the fibrinpolymerization is monitored over time to provide a time-dependentmeasurement profile.
 67. The method of claim 66, wherein an endpoint isextracted from the time-dependent measurement profile.
 68. The method ofclaim 67, wherein the endpoint is normalized by using a model.
 69. Themethod of claim 68, wherein the model is a ratio or difference of theendpoint compared to an endpoint from a time-dependent measurementprofile for a known sample.
 70. The method of claim 69, wherein theendpoint is initiation of clot formation, overall change in the profile,or slope of the profile after initiation of clot formation.
 71. Themethod according to claim 66, wherein at least two time-dependent fibrinpolymerization profiles are obtained, an additional profile beingobtained for a known sample from computer memory or by adding saidactivator at at least one concentration to a known sample and monitoringthe formation of fibrin polymerization over time.
 72. The methodaccording to claim 71, wherein at least one parameter from eachtime-dependent fibrin polymerization profile having varying activatorconcentrations is determined and a concentration at which the at leastone parameter of said sample being tested deviates from normal isdetermined.
 73. The method according to claim 67, wherein the endpointis time index or value of the minimum of the first derivative, the timeindex or value for the minimum or maximum of the second derivative, orthe overall magnitude of change.
 74. The method according to claim 66,wherein the rate or acceleration of fibrin polymerization is determinedfrom the time-dependent measurement profile, wherein said rate oracceleration is compared to rate or acceleration at the same activatorconcentration for a known sample and/or the rate or acceleration of thetest sample at a different activator concentration.
 75. The method ofclaim 63, wherein a fibrin polymerization profile is obtained with andwithout a protein C activator.
 76. The method of claim 75, wherein afibrin polymerization profile is obtained at multiple concentrations ofsaid activator which triggers thrombin explosion.
 77. The method ofclaim 76, wherein a fibrin polymerization profile is obtained atmultiple concentrations for a known sample.
 78. A method for detectingdefects in the propagation and/or amplification phase in the coagulationsystem of a test sample, comprising: providing a sample to be tested;adding an activator capable of triggering a thrombin explosion that isdependent on the propagation phase and/or amplification loops of thecoagulation system in the test sample; measuring fibrin polymerization;and detecting defects of regulation or modulation in the propagationphase and/or amplification loops in the coagulation system of the testsample based on the measured fibrin polymerization.
 79. The methodaccording to claim 78, wherein all or part of said time-dependentprofile is compared to all or part of a time-dependent profile for aknown sample.
 80. The method according to claim 79, wherein part of saidprofile is compared, said part of said profile including one or more ofinitiation of clot formation, overall change in profile, slope ofprofile after initiation of clot formation and acceleration at the timeof clot initiation.
 81. The method according to claim 79, wherein atleast two time-dependent fibrin polymerization profiles are obtained, anadditional profile being obtained for a known sample from computermemory or by adding said activator at at least one concentration to aknown sample and monitoring the formation of fibrin polymerization overtime.
 82. The method according to claim 81, wherein at least twotime-dependent fibrin polymerization profiles are obtained, one profilefor said test sample at a first activator concentration, and at leastone additional profile for said test sample at a second activatorconcentration and/or one or more profiles for a known sample at one ormore activator concentrations.
 83. The method according to claim 78,wherein the activator comprises tissue factor.
 84. The method accordingto claim 81, wherein at least one parameter from each time-dependentfibrin polymerization profile having varying activator concentrations isdetermined and a concentration at which the at least one parameter ofsaid sample being tested deviates from normal is determined.
 85. Themethod according to claim 84, wherein said at least one parameter istime index and value of the minimum of the first derivative, the timeindex and value for the minimum and maximum of the second derivative andthe overall magnitude of change.
 86. The method according to claim 82,wherein part of each fibrin polymerization profile is compared to a samepart of a profile for a known sample.
 87. The method according to claim86, wherein said part is one or more of a time index of the minimum ofthe first derivative, the value of the minimum of the first derivative,the time index for the minimum of the second derivative, the value forthe minimum of the second derivative, the time index of the maximum ofthe second derivative, the value of the maximum of the secondderivative, and the overall magnitude of change.
 88. The methodaccording to claim 88, wherein said part is rate or acceleration offibrin polymerization, wherein said rate or acceleration is compared torate or acceleration at the same activator concentration for said knownsample.
 89. The method according to claim 88, wherein a difference orratio of said parameters for said test sample and said normal sample aredetermined.
 90. The method according to claim 89, wherein said parameteris clot time and a ratio of clot times at different activatorconcentrations is determined.
 91. The method according to claim 78,wherein one or more parameters of said time-dependent fibrinpolymerization profile are compared to the same one or more parametersfor a normal sample, in order to determine whether said patient ishypercoagulable, normal or hypocoagulable.
 92. The method according toclaim 84, wherein said at least one parameter includes at least one oftime of initiation of clot formation, rate of clot formation, maximumacceleration of clot formation, turbidity at a predetermined timeperiod, and total change in turbidity.
 93. The method according to claim92 wherein said one or more parameters are measures of defects in thethrombin propagation and/or amplification phases.
 94. The methodaccording to claim 92, wherein a ratio of said at least one parameterfor said test sample to the same parameter for a normal sample isdetermined.
 95. The method according to claim 94, wherein said ratio isdetermined for multiple concentrations of activator.
 96. The methodaccording to claim 95, wherein a concentration at which said ratiodeparts from 1, or a range around 1, is determined.
 97. The methodaccording to claim 78, wherein an activator of one or more anticoagulantpathways is added.
 98. The method according to claim 97, wherein anactivator of protein C is added.
 99. The method according to claim 98,wherein the protein C activator is thrombomodulin.
 100. The methodaccording to claim 99, wherein a fibrin polymerization profile isobtained with and without said thrombomodulin.
 101. The method accordingto claim 78, wherein multiple concentrations of said activator are usedfor providing corresponding multiple time-dependent measurementprofiles, and multiple concentrations of activator of a known sample areused for providing corresponding multiple time-dependent known samplemeasurement profiles, and ratios of one or more parameters of themeasurement profiles of the known and test sample are compared.
 102. Themethod according to claim 101, wherein the one or more parameters at theone or more concentrations of said activator can be compared in thepresence or absence of a modulator of one or more anticoagulantpathways.
 103. The method according to claim 78, wherein one or moreparameters at multiple concentrations of said activator are determinedand results are compared.
 104. The method according to claim 101,wherein any concentration of said activator can be compared in thepresence or absence of a modulator of one or more anticoagulantpathways.
 105. The method according to claim 104, wherein the activatoris tissue factor and the modulator is thrombomodulin.
 106. The methodaccording to claim 78, wherein the activator comprises tissue factor andphospholipids.
 107. The method according to claim 78, wherein a metalsalt is added as part of the activator or separately therefrom, whichmetal salt dissociates into a metal divalent cation when added to thetest sample.
 108. The method according to claim 107, wherein thedivalent metal cation is magnesium, calcium or manganese.
 109. Themethod of claim 107, wherein the metal salt is a halide of magnesium,calcium or manganese.
 110. The method of claim 78, wherein the activatorcomprises purified or recombinant tissue factor.
 111. The method ofclaim 110, wherein the activator comprises homogenized brain tissue.112. The method of claim 78, further comprising adding phopholipidstogether with or separately from the activator.
 113. The method of claim78, further comprising adding buffers and/or stabilizers to the testsample.
 114. The method of claim 78, wherein the test sample is apatient plasma sample.
 115. The method of claim 79, wherein the knownsample is a normal sample.
 116. The method of claim 78, wherein the timedependent measurement profile is an optical absorbance or transmittanceprofile provided on an automated analyzer.
 117. The method of claim 116,wherein a light beam having a wavelength in the visible spectrum isdirected through a container holding the test sample and activator, andlight absorbed or transmitted is monitored to form the time dependentmeasurement profile.
 118. The method of claim 78, wherein the activatorcomprises tissue factor sufficiently diluted so as to allowdetermination of any of hypercoagulable, normal or hypcoagulabledepending upon the condition of the patient.
 119. The method of claim78, wherein a part of the time dependent measurement profile other thanclot time is compared to the same part of a time dependent measurementprofile for a known sample.
 120. The method of claim 78, wherein defectsin formation of intrinsic tenase complex are detected.
 121. The methodof claim 78, wherein one or more endpoints from the time-dependentmeasurement profile are calculated, the endpoints selected from the timeof clot initiation and the rate of polymerization.
 122. The method ofclaim 121, wherein at least one parameter selected from the firstderivative of the time dependent measurement profile, the secondderivative of the time dependent measurement profile, the minimum of thefirst and/or second derivative, or the maximum of the first and/orsecond derivative are calculated with respect to value and/or the timeassociated time index.
 123. The method of claim 122, wherein the atleast one parameter is compared to the same at least one parameter for aknown sample.
 124. The method of claim 122, wherein a first ratio iscalculated for the at least one parameter at two differentconcentrations of the activator.
 125. The method of claim 124, wherein asecond ratio is calculated of said first ratio at the two differentactivator concentrations relative to a first ratio calculated for aknown sample at two different activator concentrations.
 126. The methodof claim 125, wherein a third ratio is calculated of said second ratioat a first reagent formulation and said second ratio at a second reagentformulation.
 127. The method of claim 126, wherein the first reagentformulation comprises a coagulation activator and the second reagentformulation comprises a coagulation activator and an activator of ananticoagulant pathway.
 128. The method of claim 127, wherein the firstreagent comprises tissue factor and the second reagent comprises tissuefactor and thrombomodulin.
 129. The method of claim 125, wherein afourth ratio is calculated of said second ratio calculated for oneendpoint relative to said second ratio calculated for a differentendpoint.
 130. The method of claim 129, wherein one of the endpoints isclot time and the other is the minimum of the first derivative.
 131. Themethod of claim 78, wherein sample is whole blood or platelet richplasma.
 132. The method of claim 78, further comprising adding vesiclesto the test sample.
 133. The method of claim 132, wherein the vesiclescomprise platelets, cellular debris, lipids or platelet microparticles.134. The method of claim 78, further comprising adding a protein Cactivator to the test sample.
 135. The method according to claim 134,wherein the protein C activator is purified human thrombomodulin,purified non-human mammalian thrombomodulin, soluble or membraneassociated thrombomodulin, native thrombomodulin or thrombomodulinreconstituted with phospholipids, partially or fully glycolsylatedthrombomodulin or fully deglycosylated thrombomodulin.
 136. The methodof claim 78, wherein the activator comprises recombinant or purifiedtissue factor, truncated tissue factor, or cells expressing tissuefactor on their surface.
 137. A method for determining whether a patientis hypercoagulable, normal or hypocoagulable, comprising: providing asample to be tested from a patient; adding less than 11 picomolarconcentration of tissue factor to said sample, said tissue factorgenerating intrinsic dependent fibrin polymerization in said sample;measuring formation of the fibrin polymerization; and determiningwhether said patient is hypercoagulable, normal or hypocoagulable basedon said measured fibrin polymerization.
 138. The method according toclaim 137, wherein said fibrin polymerization is measured over time soas to derive a time-dependent fibrin polymerization profile.
 139. Themethod according to claim 138, wherein one or more parameters of saidfibrin polymerization profile are compared to the same parameters of afibrin polymerization profile for a normal sample or for the same testsample where the activator or the activator concentration is changed.140. The method according to claim 139, wherein said one or moreparameters do not include clot time.
 141. The method of claim 139,wherein the one or more parameters are determined or calculated based oninformation in the time dependent measurement profiles which are afterinitiation of clot formation.
 142. The method according to claim 141,wherein said one or more parameters include the rate of fibrinpolymerization.
 143. The method according to claim 137, wherein saidsample comprises endogenous or exogenous fibrinogen.
 144. The methodaccording to claim 143, wherein the measurement of fibrin polymerizationis performed in the absence of a chromogenic substrate in the testsample.
 145. The method according to claim 137, wherein the test sampleis a non-diluted native plasma sample and the activator added theretocomprises tissue factor.
 146. The method according to claim 145, furthercomprising adding phosphatidylcholine, phosphatidylethanolamine and/orphosphatidylserine as part of the activator or separately therefrom.147. The method according to claim 137, wherein at least a portion ofsaid time-dependent profile or a value derived therefrom is compared tothe same portion or value for a known sample.
 148. The method accordingto claim 147, wherein part of said profile is compared, said part ofsaid profile including one or more of initiation of clot formation,overall change in profile, and slope of profile after initiation of clotformation.
 149. The method according to claim 147, wherein at least twotime-dependent fibrin polymerization profiles are obtained, anadditional profile being obtained for a known sample from computermemory or by adding said activator at at least one concentration to aknown sample and monitoring the formation of fibrin polymerization overtime.
 150. The method according to claim 149, wherein at least twotime-dependent fibrin polymerization profiles are obtained, one profilefor said test sample at a first activator concentration, and at leastone additional profile for said test sample at a second activatorconcentration and/or one or more profiles for a known sample at one ormore activator concentrations.
 151. The method according to claim 137,wherein the activator comprises tissue factor.
 152. The method accordingto claim 149, wherein at least one parameter from each time-dependentfibrin polymerization profile at a different activator concentration isdetermined and a concentration at which the at least one parameter ofsaid sample being tested deviates from normal, or a range around normal,is determined.
 153. The method according to claim 152, wherein saidparameter is one or more of a time index of the minimum of the firstderivative, the value of the minimum of the first derivative, the timeindex for the minimum of the second derivative, the value for theminimum of the second derivative, the time index of the maximum of thesecond derivative, the value of the maximum of the second derivative,and the overall magnitude of change.
 154. The method according to claim152, wherein said parameter is rate or acceleration of fibrinpolymerization, wherein said rate or acceleration is compared to rate oracceleration at the same activator concentration for said known sample.155. The method according to claim 152, wherein a difference or ratio ofsaid parameters for said test sample and said known sample aredetermined.
 156. The method according to claim 152 wherein said at leastone parameter is a measure of defects in the thrombin propagation andamplification phases.
 157. The method according to claim 155, whereinsaid ratio is determined for multiple concentrations of activator. 158.The method according to claim 155, wherein a concentration at which saidratio departs from 1, or a range around 1, is determined.
 159. Themethod according to claim 137, further comprising adding an activator ofone or more anticoagulant pathways.
 160. The method according to claim159, wherein an activator of protein C is added.
 161. The methodaccording to claim 160, wherein the protein C activator isthrombomodulin.
 162. The method according to claim 161, wherein a fibrinpolymerization profile is obtained with and without said thrombomodulin.163. The method according to claim 137, wherein multiple concentrationsof said activator are used for providing corresponding multipletime-dependent measurement profiles, and multiple concentrations ofactivator of a known sample are used for providing correspondingmultiple time-dependent known sample measurement profiles, and ratios ofone or more parameters of the measurement profiles of the known and testsample are compared.
 164. The method according to claim 137, wherein anyconcentration of said activator can be compared in the presence orabsence of a modulator of one or more anticoagulant pathways.
 165. Themethod according to claim 137, wherein a metal salt is added as part ofthe activator or separately therefrom, which metal salt dissociates intoa metal divalent cation when added to the test sample.
 166. The methodaccording to claim 165, wherein the divalent metal cation is magnesium,calcium or manganese.
 167. The method of claim 165, wherein the metalsalt is a halide of magnesium, calcium or manganese.
 168. The method ofclaim 137, wherein the activator comprises purified or recombinanttissue factor.
 169. The method of claim 168, wherein the activatorcomprises homogenized brain tissue.
 170. The method of claim 137,further comprising adding phospholipids together with or separately fromthe activator.
 171. The method of claim 137, further comprising addingbuffers and/or stabilizers to the test sample.
 172. The method of claim137, wherein the time dependent measurement profile is an opticalabsorbance or transmittance profile provided on an automated analyzer.173. The method of claim 137, wherein the activator comprises tissuefactor sufficiently diluted so as to allow determination of any ofhypercoagulable, normal or hypcoagulable depending upon the condition ofthe patient.
 174. The method of claim 137, wherein a part of the timedependent measurement profile other than clot time is compared to thesame part of a time dependent measurement profile for a known sample.175. The method of claim 137, wherein defects in formation of intrinsictenase complex are detected.
 176. The method of claim 137, wherein afirst ratio is calculated for the at least one parameter at twodifferent concentrations of the activator.
 177. The method of claim 176,wherein a second ratio is calculated of said first ratio at the twodifferent activator concentrations relative to a first ratio calculatedfor a known sample at two different activator concentrations.
 178. Themethod of claim 177, wherein a third ratio is calculated of said secondratio at a first reagent formulation and said second ratio at a secondreagent formulation.
 179. The method of claim 178, wherein the firstreagent formulation comprises a coagulation activator and the secondreagent formulation comprises a coagulation activator and an activatorof an anticoagulant pathway.
 180. The method of claim 179, wherein thefirst reagent comprises tissue factor and the second reagent comprisestissue factor and thrombomodulin.
 181. The method of claim 177, whereina fourth ratio is calculated of said second ratio calculated for oneendpoint relative to said second ratio calculated for a differentendpoint.
 182. The method of claim 181, wherein one of the endpoints isclot time and the other is the minimum of the first derivative.
 183. Themethod of claim 137, further comprising adding vesicles to the testsample.
 184. The method of claim 182, wherein the vesicles compriseplatelets, cellular debris, phospholipid vericles or plateletmicroparticles.
 185. The method of claim 137, further comprising addinga protein C activator to the test sample.
 186. The method according toclaim 185, wherein the protein C activator is purified humanthrombomodulin, purified non-human mammalian thrombomodulin, soluble ormembrane associated thrombomodulin, native thrombomodulin orthrombomodulin reconstituted with phospholipids, partially or fullyglycolsylated thrombomodulin or fully deglycosylated thrombomodulin.187. The method of claim 137, wherein the activator comprisesrecombinant or purified tissue factor, truncated tissue factor, or cellsexpressing tissue factor on their surface.
 188. A method for monitoringan antithrombotic or procoagulant pharmaceutical therapy, comprising:providing a first test sample from a patient; adding an activator tosaid test sample in order to trigger a thrombin explosion dependent uponthe propagation phase and amplification loops of the coagulation systemin the test sample; measuring fibrin polymerization due at least in partto said thrombin explosion; determining whether the patient ishypocoagulable, normal or hypercoagulable, or providing a baseline; ifthe patient is hypercoagulable or hypocoagulable, adminstering one ormore antithrombotic or procoagulant pharmaceuticals to said patient;providing at least one additional sample from said patient at a timeafter administration of the pharmaceutical; adding said activator tosaid at least one additional sample in order to trigger a thrombinexplosion dependent upon the propagation phase and amplification loopsof the coagulation system in the test sample; measuring fibrinpolymerization in said second sample due at least in part to saidthrombin explosion; determining whether the second patient sample ishypocoagulable, normal or hypercoagulable, or determining a change frombaseline; and determining the effectiveness of the pharmaceuticaltherapy based on any changes in the hypocoagulability orhypercoagulability from the first test sample, or any changes frombaseline.
 189. The method of claim 188, further comprising addingvesicles to the test sample.
 190. The method of claim 189, wherein thevesicles comprise platelets, cellular debris, phospholipid vesicles orplatelet microparticles.
 191. The method of claim 188, wherein anactivator of protein C is added to cause the fibrin polymerization to besensitive to the protein C pathway.
 192. The method according to claim191, wherein the protein C activator is purified human thrombomodulin,purified non-human mammalian thrombomodulin, soluble or membraneassociated thrombomodulin, native thrombomodulin or thrombomodulinreconstituted with phospholipids, partially or fully glycolsylatedthrombomodulin or fully deglycosylated thrombomodulin.
 193. The methodof claim 188, wherein the activator comprises recombinant or purifiedtissue factor, truncated tissue factor, or cells expressing tissuefactor on their surface.
 194. The method of claim 188, wherein thefibrin polymerization is monitored over time to provide a time-dependentmeasurement profile.
 195. The method of claim 194, wherein an endpointis extracted from the time-dependent measurement profile.
 196. Themethod of claim 195, wherein the endpoint is normalized by using amodel.
 197. The method of claim 196, wherein the model is a ratio ordifference of the endpoint compared to an endpoint from a time-dependentmeasurement profile for a known sample.
 198. The method of claim 197,wherein the endpoint is initiation of clot formation, overall change inthe profile, or slope of the profile after initiation of clot formation.199. The method according to claim 194, wherein at least twotime-dependent fibrin polymerization profiles are obtained, anadditional profile being obtained for a known sample from computermemory or by adding said activator at at least one concentration to aknown sample and monitoring the formation of fibrin polymerization overtime.
 200. The method according to claim 199, wherein at least oneparameter from each time-dependent fibrin polymerization profile havingvarying activator concentrations is determined and a concentration atwhich the at least one parameter of said sample being tested deviatesfrom normal is determined.
 201. The method according to claim 195,wherein the endpoint is time index or value of the minimum of the firstderivative, the time index or value for the minimum or maximum of thesecond derivative, or the overall magnitude of change.
 202. The methodaccording to claim 194, wherein the rate or acceleration of fibrinpolymerization is determined from the time-dependent measurementprofile, wherein said rate or acceleration is compared to rate oracceleration at the same activator concentration for a known sampleand/or the rate or acceleration of the test sample at a differentactivator concentration.
 203. The method of claim 191, wherein a fibrinpolymerization profile is obtained with and without a protein Cactivator.
 204. The method of claim 203, wherein a fibrin polymerizationprofile is obtained at multiple concentrations of said activator whichtriggers thrombin explosion.
 205. The method of claim 204, wherein afibrin polymerization profile is obtained at multiple concentrations fora known sample.
 206. A method for evaluating the efficacy of anantithrombotic or procoagulant pharmaceutical, comprising: providing afirst test sample from a human or non-human mammal; adding an activatorto said first test sample in order to trigger a thrombin explosiondependent upon the propagation phase and amplification loops of thecoagulation system in the test sample; measuring fibrin polymerizationin the first test sample due at least in part to said thrombinexplosion; determining whether the sample is hypocoagulable, normal orhypercoagulable, or providing a baseline; administering one or moreantithrombotic or procoagulant pharmaceuticals to the mammal; providingat least one additional sample from the mammal at a time afteradministration of the pharmaceutical; adding said activator to said atleast one additional sample in order to trigger a thrombin explosiondependent upon the propagation phase and amplification loops of thecoagulation system in the test sample; measuring fibrin polymerizationin said at least one additional sample due at least in part to saidthrombin explosion; determining the degree of hypocoagulability orhypercoagulability of the second mammalian sample, or a change frombaseline; and determining the efficacy of the pharmaceutical based onany changes in the hypocoagulability or hypercoagulability from thefirst test sample, or any changes from baseline.
 207. The method ofclaim 206, further comprising adding vesicles to the test sample. 208.The method of claim 207, wherein the vesicles comprise platelets,cellular debris, phospholipid vesicles or platelet microparticles. 209.The method of claim 206, wherein an activator of protein C is added tocause the fibrin polymerization to be sensitive to the protein Cpathway.
 210. The method according to claim 209, wherein the protein Cactivator is purified human thrombomodulin, purified non-human mammalianthrombomodulin, soluble or membrane associated thrombomodulin, nativethrombomodulin or thrombomodulin reconstituted with phospholipids,partially or fully glycolsylated thrombomodulin or fully deglycosylatedthrombomodulin.
 211. The method of claim 206, wherein the activatorcomprises recombinant or purified tissue factor, truncated tissuefactor, or cells expressing tissue factor on their surface.
 212. Themethod of claim 206, wherein the fibrin polymerization is monitored overtime to provide a time-dependent measurement profile.
 213. The method ofclaim 212, wherein an endpoint is extracted from the time-dependentmeasurement profile.
 214. The method of claim 213, wherein the endpointis normalized by using a model.
 215. The method of claim 214, whereinthe model is a ratio or difference of the endpoint compared to anendpoint from a time-dependent measurement profile for a known sample.216. The method of claim 215, wherein the endpoint is initiation of clotformation, overall change in the profile, or slope of the profile afterinitiation of clot formation.
 217. The method according to claim 212,wherein at least two time-dependent fibrin polymerization profiles areobtained, an additional profile being obtained for a known sample fromcomputer memory or by adding said activator at at least oneconcentration to a known sample and monitoring the formation of fibrinpolymerization over time.
 218. The method according to claim 217,wherein at least one parameter from each time-dependent fibrinpolymerization profile having varying activator concentrations isdetermined and a concentration at which the at least one parameter ofsaid sample being tested deviates from normal is determined.
 219. Themethod according to claim 213, wherein the endpoint is time index orvalue of the minimum of the first derivative, the time index or valuefor the minimum or maximum of the second derivative, or the overallmagnitude of change.
 220. The method according to claim 212, wherein therate or acceleration of fibrin polymerization is determined from thetime-dependent measurement profile, wherein said rate or acceleration iscompared to rate or acceleration at the same activator concentration fora known sample and/or the rate or acceleration of the test sample at adifferent activator concentration.
 221. The method of claim 209, whereina fibrin polymerization profile is obtained with and without a protein Cactivator.
 222. The method of claim 221, wherein a fibrin polymerizationprofile is obtained at multiple concentrations of said activator whichtriggers thrombin explosion.
 223. The method of claim 222, wherein afibrin polymerization profile is obtained at multiple concentrations fora known sample.
 224. The method of claim 207, wherein a part of the timedependent profile for each sample is compared to the same part of a timedependent measurement profile for a known sample.
 225. A methodcomprising: providing a plasma or whole blood sample from a firstpatient; adding one or more reagents for activating coagulation, and ametal cation or metal salt which dissociates into a metal cation, andvesicles; determining that the patient is hypercoagulable orhypocoagulable; providing a plasma or whole blood sample from a secondpatient; adding the one or more reagents comprising the same coagulationactivator, metal cation or metal salt, and vesicles as in step (b) tothe second patient sample; determining that the second patient is theother of hypocoagulable or hypercoagulable opposite to the firstpatient.
 226. A method for assessing the hemostatic potential of asample comprising: a. providing a sample to be tested; b. adding acoagulation activator to the sample; c. generating a time dependentmeasurment profile; and d. assessing the hemostatic potential of thesample from the time dependent measurement profile.
 227. The method ofclaim 226, further comprising determining whether the sample ishypocoagulable, normal or hypercoagulable based on the assessedhemostatic potential.
 228. The method of claim 226, further comprisingdetermining whether a patient from whom the sample was taken has athrombotic or hemorhagic tendency.
 229. The method according to claim226, wherein all or part of said time-dependent profile is compared toall or part of a time-dependent profile for a known sample.
 230. Themethod according to claim 229, wherein part of said profile is compared,said part of said profile including one or more of initiation of clotformation, overall change in profile, slope of profile after initiationof clot formation, and acceleration at the time of clot initiation. 231.The method according to claim 229, wherein at least two time-dependentfibrin polymerization profiles are obtained, an additional profile beingobtained for a known sample from computer memory or by adding saidactivator at at least one concentration to a known sample and monitoringthe formation of fibrin polymerization over time.
 232. The methodaccording to claim 231, wherein at least two time-dependent fibrinpolymerization profiles are obtained, one profile for said test sampleat a first activator concentration, and at least one additional profilefor said test sample at a second activator concentration and/or one ormore profiles for a known sample at one or more activatorconcentrations.
 233. The method according to claim 226, wherein theactivator comprises tissue factor.
 234. The method according to claim231, wherein at least one parameter from each time-dependent fibrinpolymerization profile having varying activator concentrations isdetermined and a concentration at which the at least one parameter ofsaid sample being tested deviates from normal is determined.
 235. Themethod according to claim 234, wherein said at least one parameter isselected from time index and value of the minimum of the firstderivative, the time index and value for the minimum and maximum of thesecond derivative and the overall magnitude of change.
 236. The methodaccording to claim 232, wherein part of each fibrin polymerizationprofile is compared to a same part of a profile for a known sample. 237.The method according to claim 236, wherein said part is one or more of atime index of the minimum of the first derivative, the value of theminimum of the first derivative, the time index for the minimum of thesecond derivative, the value for the minimum of the second derivative,the time index of the maximum of the second derivative, the value of themaximum of the second derivative, and the overall magnitude of change.238. The method according to claim 236, wherein said part is rate oracceleration of fibrin polymerization, wherein said rate or accelerationis compared to rate or acceleration at the same activator concentrationfor said known sample.
 239. The method according to claim 236, wherein adifference or ratio of said parameters for said test sample and saidnormal sample are determined.
 240. A method comprising: providing a testsample from the patient; initiating coagulation in the sample in thepresence of a coagulation activator and optionally an activator of ananticoagulant pathway, the coagulation activator added to the sample inan amount which will result in intrinsic tenase-dependent fibrinpolymerization; monitoring formation of said intrinsic tenase-dependentfibrin polymerization over time so as to derive a time-dependentprofile; looking at an endpoint from the time-dependent profile toassess the hemostatic potential of the test sample.
 241. The method ofclaim 240, further comprising: repeating steps a) to d) but changing theconcentration of the coagulation activator, changing the concentrationof the activator of an anticoagulant pathway, and/or changing theendpoint.
 242. The method of claim 241, wherein step e) is performedwhen the first patient sample is hypercoagulable or hypocogulable. 243.The method of claim 242, wherein step e) is performed when the firstpatient sample is mildly hypercoagulable or hypocoagulable.
 244. Themethod of claim 240 performed on an automated coagulation analyzer. 245.The method of claim 244, wherein the time dependent profile is providedby monitoring light absorbance or transmittance through a cuvette. 246.The method of claim 241, wherein the coagulation activator is tissuefactor, the anticoagulant pathway activator is thrombomodulin, and theendpoint is selected from a time index of the minimum of the firstderivative, the value of the minimum of the first derivative, the timeindex for the minimum of the second derivative, the value for theminimum of the second derivative, the time index of the maximum of thesecond derivative, the value of the maximum of the second derivative,and the overall magnitude of change.
 247. The method of claim 241,wherein the endpoint is other than clot time.
 248. The method of claim241, wherein more than one of the concentration of the coagulationactivator, the concentration of the activator of an anticoagulantpathway, and the endpoint are altered in step e).
 249. The method ofclaim 241, wherein the endpoint is initiation of clot formation, overallchange in the time dependent profile, slope of the profile afterinitiation of clot formation, and/or acceleration at the time of clotinitiation.
 250. The method of claim 240, wherein the endpoint is avariable within a curve fit function.
 251. The method of claim 188,wherein the fibrin polymerization measurement is used to adjust thepatient's therapy to result in a fibrin polymerization profileapproximating normal.
 252. A method for assessing the hemostaticpotential of a sample, comprising: adding to a sample a coagulationactivator, phospholipid vesicles, metal ions or metal salt if the sampleis citrated, and optionally an activator of an anticoagulant pathway;monitoring the polymerization of fibrin in the sample; and assessing thehemostatic potential of the sample based on the kinetics of the fibrinpolymerization; wherein the coagulation activator is tissue factorsufficiently diluted so as to result in an approximately 0.75 to 3.0pico molar concentration range when the reagent is mixed with thesample.